Sarcocystis cruzi: First Molecular Identification from Cattle in Iran.

Sarcocystis is a genus of cyst-forming parasites infecting both animals and human. This study aimed to isolate coccidian tissue cysts from muscle of infected animals by a simple method in addition to molecular identification of Sarcocystis cruzi from the samples. The samples were obtained from commercial source in Babol, Northern Iran. Five grams of calf muscle was cut into 2-3 pieces in 30 ml of phosphate-buffered saline containing 0.1% Tween 80 and homogenized with IKA T25, DIGITAL ULTRA-TURRAX. The homogenate was filtrated twice and used for microscopy examination and molecular analysis. Polymerase chain reaction (PCR) and partial sequence analysis of the 18S ribosomal gene were used to identify the Sarcocystis species. Giemsa stain of the filtrated calf muscle samples showed that the sample had ellipse to around tissue cysts contained crescent-shaped bodies. The PCR of the 18SrDNA yielded an 1100 bp DNA band on agarose gel and sequence analysis of the DNA confirmed the isolate as S. cruzi. The sequence was deposited in GenBank by Accession No.KC508514. This is the first molecular identification of an isolate of S. cruzi in Iran.

. This may affect the concrete detection of the species because the appearance of sarcocysts may change in accordance to the location and developmental stage of cysts and other conditions of parasitized cell. Therefore molecular studies have been suggested to confirm morphological species identification (6)(7)(8). Also, analysis of DNA sequences is useful to identify Sarcocystis species using the variable regions of the 18S rRNA gene which have been reported is a valuable targets for the identification and characterization of different species, even from the same genus (3). In Iran, the prevalence of Sarcocystis infections has been reported to be high in domestic animals including cattle, sheep, goats, and camels (9-11) but a few studies have identified the involving species (12). In the current study, the first molecular identification of S. cruzi from a calf muscle sample in the north of Iran is documented.

Cyst isolation from skeletal calf muscles
Muscle samples were obtained from commercial source in Babol, Northern Iran. 5 grams of calf muscle was cut into 2-3 pieces. The sample was added to 30 ml of phosphate-buffered saline containing 0.1% Tween 80(PBS-Tween) pH 7.4(Sigma), in a 50 ml conical flask (14). The resultant was homogenized with IKA T25, DIGITAL ULTRA-TURRAX (Germany) at 8000 RPM for 2 min. The homogenate was then filtered through two layers of gauze twice. The filtrate was collected and saved for further assessments including microscopy examination, cultivation in the laboratory animals and molecular analysis.

Microscopy examination
A smear was prepared from the filtrated calf muscle suspension. The smear was air dried and fixed with methanol for 30 second and then stained with Giemsa. The maintenance and concern of laboratory animals complied with the guidelines for the human use of laboratory animals. 2 ml of the filtrate muscle sample was inoculated to four mice peritoneal (0.5 ml per one mouse) (NMRI strain).
Two experimentally infected mice were held for one month and two of them were killed with Ethyl ether on day seven. Then, 5 ml sterile PBS was injected to mice peritoneal and then mice peritoneal fluid was collected. The peritoneal fluid was centrifuged 5 minutes at 2000 rpm. The supernatant was discarded and a smear was prepared from the pellet and stained with Giemsa.

Histology
Meat sample fixed in 10% formalin were processed as usual, sectioned at 7 µm and stained with haematoxylin and eosin (HE). The sections were examined with a light microscope at 10 and 40ҳ magnifications.

Results
Five calves muscle samples were obtained.
Examination of the filtrated calf muscle samples using Giemsa staining method showed that all meat had ellipse to around tissue cysts contained crescent-shaped bodies and some free zoites. The cysts had different sizes (Figure 1.A). Moreover, microscopical cysts were observed in the histological sections of the calves muscle. No inflammatory reaction was found around the cysts in the tissue (Figure 1.B).

Cyst -forming coccidian parasites such as
Sarcocystis spp. has ubiquitous distribution globally.
One of important potential source of these infections is meat. Many researchers have been focused on isolation of the tissue cysts as a means to teach food safety, to introduce some basic concepts about infectious disease and cell biology (13). Several methods have been developed to isolate the cystsforming parasites. The first report on separation of T. gondii muscular cyst was in 1960. In this method, a solution containing acid and pepsin was used to recover T. gondii from muscular tissues but numbers of infective organisms were too low (16). In subsequent assays Percoll gradient (17) and Dextran (18) were used to obtain more cysts and viable bradyzoites. These methods are relatively laborintensive and need several materials. In the current study a method was developed to separate coccidian tissue cysts-forming parasites from skeletal muscles of domestic animals using a homogenizer.
Generally, this apparatus has been used for batch homogenizing of cell tissues, emulsifying suspen-  miescheriana was reported from a wild boar (12) and S. fusiformis was identified in water buffalo (19). But, there is only one report of S. cruzi from Iran which identified in two infected calves based on clinical, morphological and pathological criteria after experimental infection in two puppies and consequently in a calf (20). However, S. cruzi has a global distribution and mainly reported from cattle and water buffalos of different countries throughout the world (2,21).
In conclusion, the method described in the current study would be useful in separation of tissue cysts of coccidian parasites from meat to perform further examinations such as molecular characterization of the Sarcocystis species from domestic and wild animals.